The present invention is directed to methods of treating inflammation associated with rhinitis or sinusitis by intranasally delivering peptidases to a patient. The invention also encompasses therapeutic packages in which a peptidase is preloaded in a device designed for intranasally delivering drug.
Rhinitis, an inflammation of the nasal mucosal membrane, is characterized by sneezing, rhinorrhea, nasal congestion, and increased nasal secretion. It is often accompanied by sinusitis, an inflammation of the sinuses. When these conditions persist for a period of more than three weeks, they are termed xe2x80x9cchronic.xe2x80x9d More than 37 million Americans, particularly those with allergies or asthma, suffer from these conditions, making them the most common chronic medical problems in the United States.
Chronic xe2x80x9crhinosinusitisxe2x80x9d or sinusitis is difficult to treat successfully. In general, treatment consists of a combination of antibiotics and decongestants or antihistamines. In addition, steroid nasal sprays are commonly used to reduce inflammation. For patients with severe chronic sinusitis, oral steroids, such as prednisone, may also be prescribed. However, the long-term safety of steroid administration, especially in children, is not fully understood and oral steroids often have significant side effects. When drug therapy fails, surgery is usually the only alternative.
The mucosal tissue lining the nasal and sinus passages is densely packed with sensory neurons (Alving, et al., Cell Tissue Res. 264:529-538 (1991); Saria, et al., Am. Rev. Respir. Dis. 147:1330-1335 (1988)). When activated, these neurons release a variety of bioactive peptides that contribute to inflammation by causing vasodilation, stimulating mucosal gland secretion, and promoting infiltration by inflammatory mast cells, lymphocytes and eosinophils (Stead, et al, Immunol. Rev. 10:333-359 (1987); Mygind, et al., Eur. J Respir. Dis. 64(S128):1-379 (1983)). Included among the released bioactive peptides are substance P, calcitonin-gene related peptide, neuropeptide Y and vasoactive intestinal peptide (Lundblad, et al., Acta. Physiol. Scand. 529:1-42 (1984)). Means for controlling the activity of these peptides should provide an effective treatment for the inflammation associated with both rhinitis and sinusitis.
The present invention stems from the discovery that dipeptidyl peptidase IV, an exopeptidase that cleaves Xaa-Pro dipeptides from the N-terminus of polypeptides, is present in human nasal mucosa at levels that are inversely related to inflammation. Thus, low levels of dipeptidyl peptidase IV are associated with a high density of inflammatory cells, and high levels of dipeptidyl peptidase IV are associated with a low density of inflammatory cells. This is important because dipeptidyl peptidase IV degrades peptides that contribute to the pathophysiology of rhinitis, sinusitis and asthma (Mentlein, et al., Regul. Peptides 49:133-144 (1993); Heymann, et al, FEBS Lett. 91:360-364 (1978); Beauvais, et al., Fum. Infect. Immun. 65:3042-3047 (1997)). Among the inflammation-related peptides cleaved are NPY, SP, and desArg1 bradykinin. Based upon these observation and further experiments, the concept has been developed that the intranasal administration of dipeptidyl peptidase IV can reduce inflammation in the mucosal tissue that lines both the nasal cavity and sinuses. Other proteases that possess the same proteolytic activity should also produce a positive therapeutic effect. These proteases include quiescent cell proline dipeptidase (Underwood, et al., J. Biol. Chem. 274:34053-34058 (1999)), dipeptidyl peptidase 8 (Abbott, et al., Eur. J Biochem. 267:6140-6150 (2000)), and attractin (Duke-Cohan, et al., Proc. Nat""l. Acad. Sci. U.S.A. 95:11336-11341 (1998)).
In its first aspect, the invention is directed to a method of treating a patient for inflammation of the nasal or sinus mucosa. The method involves intranasally administering a therapeutically effective amount of a peptidase (preferably an exopeptidase) that cleaves at Xaa-Pro residues, where Xaa represents any of the 20 amino acids commonly found in animals. A xe2x80x9ctherapeutically effectivexe2x80x9d dose is defined as an amount sufficient to produce a significant reduction in inflammation as evidence by either a reduced number of inflammatory cells in mucosal tissue or by a significant improvement in one or more symptoms associated with the inflammation. For example, in the case of rhinitis or sinusitis, a therapeutically effective dose would be a sufficient amount to produce a significant reduction in sneezing, coughing, sinus-related headaches, nasal obstruction, mucosal secretion, or other discomfort associated with these conditions. Inflammatory cells include mast cells, lymphocytes and eosinophils. In general, it is expected that a therapeutically effective dose for any of the proteases used will be between 1 microgram and 1 milligram and, typically, between 5 micrograms and 500 micrograms.
The preferred peptidase for use in the method is dipeptidyl peptidase IV. Other peptidases that can be used include quiescent cell proline dipeptidase; dipeptidyl peptidase 8, and attractin. In each case, it is the human form of the peptidase that is preferred. However, peptidases from other species (e.g., that secreted by Aspergilus Fumigatus, see Examples section) may also be used provided that they have the ability to cleave at the Xaa-Pro sequence. Although the method will work for rhinitis and sinusitis caused by any disease or condition, it is expected that the most common causes will be allergies or asthma.
In another aspect, the invention is directed to a therapeutic package in which a device for intranasally delivering drug to a patient is preloaded with a solution or powder containing one or more of the peptidases described above. The invention is compatible with any intranasal delivery device (including encapsulated dosage forms) and with any of the numerous compositions that have been described for delivering drugs by means of the nasal cavity. When liquid compositions are used in the device, it is expected that peptidase will be present at a concentration of between 1 xcexcg/ml and 10 mg/ml, and more typically, at a concentration of between 10 xcexcg/ml. and 1 mg/ml.
The invention also encompasses the concept that SP is particularly important in causing inflammation in lung and nasal mucosa. Any method that reduces the local activity of this peptide should be useful in the treatment of rhinitis or sinusitis. A reduction in activity may be accomplished either using a peptidase that degrades SP (e.g., one of the peptidases described above) or by administering an agent that inhibits the binding of SP to the NK1 receptor (see Examples section).
A. Preparation of Peptidases
The present invention is directed to treatment methods which utilize peptidases that have the common characteristic of cleaving at Xaa-Pro sites. These may be purchased commercially or obtained using any of the procedures described in the relevant literature. For example, the gene corresponding to the peptidase can be isolated and used for recombinant protein production. Especially preferred peptidases, along with references relevant to their isolation and recombinant production, are: human dipeptidyl peptidase IV, shown herein as SEQ ID NO: 1 (Misumi, et al, Biochim. Biophys. Acta 15:1131 (1992); Darmoul, et al., J. Biol. Chem. 267:4824-4833 (1992); Abbott, et al., Immunogenetics 40:331-338 (1994)); human quiescent cell proline dipeptidase, shown herein as SEQ ID NO: 2 (Underwood, et al, J. Biol. Chem. 274:34053-34058 (1999)); human attractin, shown herein as SEQ ID NO: 3 (Duke-Cohan, et al., Proc. Nat""l. Acad. Sci. USA 95:11336-11341 (1998); Nagase, et al, DNA Res. 5:31-39 (1998)); and human dipeptidyl peptidase 8, shown herein as SEQ ID NO: 4 (Abbott, et al., Eur. J. Biochem. 267:6140-6150 (2000)). In addition to being made recombinantly, these proteins can be synthesized using methods that are well-known in the art.
B. Making of Pharmaceutical Compositions
Compositions for intranasally delivering peptidases can be made in accordance with methods that are standard in the art (see, e.g., Remington""s Pharmaceutical Sciences, 16th ed. A. Oslo Editor, Easton, Pa. (1980)). Enzymes will typically be prepared in admixture with conventional excipients. Suitable carriers may include, but are not limited to: water; salt solutions; alcohols; vegetable oils; polyethylene glycols; gellatin; carbohydrates such as lactose, amylose or starch; talc; hydroxymethylcellulose etc. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents such as preservatives or stabilizers. The invention is compatible with any of the numerous compositions that have been disclosed in the art for nasal delivery including those in: U.S. Pat. Nos. 6,054,462; 4,946,870; 5,897,858; and 4,476,116. The concentration of peptidase present can vary over a wide range. Typically, in liquid formulations, peptide should be present in an amount of between 1 xcexcg/ml and 10 mg/ml and, more commonly, at a concentration of between 10 xcexcg/ml and 1 mg/ml.
Treatment Methods
The present invention is directed to methods for treating inflammation present in the mucosal lining of the nasal or sinus passages. It depends upon the direct administration of sufficient peptidase to proteolytically cleave peptides known to contribute to inflammation. The total dosage of peptidase to be administered to a patient should be at least the amount required to achieve this objective as reflected by a reduction or elimination of symptoms associated with inflammation. For example, a patient being treated for rhinosinusitis should receive sufficient compound to reduce or eliminate the frequency or intensity of sinus headache, reduce coughing, congestion, sneezing, respiratory obstruction or other discomforts associated with the condition.
In general, a patient may begin by self-administering a relatively small dose of compound and then repeat administration as necessary. For example, a patient may begin by administering 0.1 mg per day and then increase the dosage upward using changes in inflammation-related symptoms as a guide. Typically, it is expected that patients will receive a daily dose of between 1 xcexcg and 1 mg per day, and, more typically, between 5 xcexcg and 500 xcexcg. Daily dosages may be provided in either a single or multiple regimen with the latter being generally preferred. These are simply guidelines, since the actual dose will be determined by the patient and their physician based upon a variety of clinical factors.
Therapeutic Packages
In addition to the pharmaceutical compositions described above, the invention includes therapeutic packages for the intranasal delivery of the compositions. A therapeutic package is comprised of a device designed for the intranasal inhalation of medication which has been preloaded with a pharmaceutical composition containing one or more of the peptidases described above. In general, spray devices are preferred, such as those disclosed in U.S. Pat. No. 6,145,703; WO 95/00195; U.S. Pat. No. 5,307,953; EP 0388651; U.S. Pat. Nos. 4,017,007; 5,301,846; 3,176,883; or 4,286,735. Devices for administering powders or nasal drops may also be used. When the pharmaceutical composition is in the form of a solution, it is expected that peptidase should generally be present at a concentration of between 1 xcexcg/ml and 10 mg/ml, and, more typically, at a concentration of between 10 xcexcg/ml and 1 mg/ml.